Method for inducing oligodendrocyte precursor cells from oct4-induced human somatic cells through direct reprogramming

ABSTRACT

Provided is a method of inducing oligodendrocyte precursor cells (OPCs) through direct reprogramming from human somatic cells into which a nucleic acid molecule encoding an Oct4 protein or Oct4 protein-treated human somatic cells. The method of inducing OPCs by treating Oct4-overexpressing human somatic cells with a low molecular weight substance may establish OPCs with high efficiency in a short period of time through direct reprogramming without via neural stem cells, and thus the OPCs are useful as a cell therapeutic agent for an intractable demyelinating disease.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/575,369 filed on Nov. 19, 2017, which is a National Phase of PCTPatent Application No. PCT/KR2016/004603 having International filingdate of May 2, 2016, which claims the benefit of priority of KoreanPatent Applications Nos. 10-2015-0069696 filed on May 19, 2015, and10-2016-0043593 filed on Apr. 8, 2016. The contents of the aboveapplications are all incorporated by reference as if fully set forthherein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a method of inducing oligodendrocyteprecursor cells (OPCs) through direct reprogramming from human somaticcells into which a nucleic acid molecule encoding an Oct4 protein hasbeen introduced or Oct4 protein-treated human somatic cells.

Due to the progression of an aging society, personalized celltherapeutic agents for healthier aging and a healthy life withoutdisease are medications essential for improving the quality of life.Multiple sclerosis known as a disease of the central nervous system is ademyelinating disease whose cause is unknown, and in severe cases, isaccompanied by sensory and motor impairment. However, there is nofundamental therapy, except treatment with medications for reducingsymptoms. Accordingly, transplantation of OPCs (OPC) that candifferentiate into oligodendrocytes enabling the generation of myelinsheaths has attracted attention as a main therapeutic method, andresearch to obtain cells which will be used for the transplantation isprogressing using embryonic stem cells and adult stem cells.

Embryonic stem cells are pluripotent cells which are able todifferentiate into all types of human cells having the ability to divideindefinitely, unlike somatic cells. Adult stem cells are multipotentcells which are able to be extracted from a patient, and as arepresentative example, neural stem cells (NSCs) are well known. NSCs,which are adult stem cells, can overcome immune rejection in thetreatment of neurological diseases, and therefore has attractedattention as a cell therapeutic agent. However, NSCs are not effectivebecause they have a considerably low ability to differentiate intooligodendrocytes, and are limited in number of cells because they shouldbe obtained from a patient's own cerebral tissue. Embryonic stem cellsalso have disadvantages to be overcome for clinical use. First, there isan ethical issue because it is necessary to destroy a fertilized embryoto obtain embryonic stem cells, and when cells differentiated from theembryonic stem cells are transplanted into a patient, immune rejectionoccurs.

Among various methods attempting to overcome such problems, a method ofdedifferentiation from differentiated cells to undifferentiated cellshas attracted attention, and dedifferentiation encompasses thegeneration of pluripotent stem cells such as embryonic stem cells usingdifferentiated cells. After induced pluripotent stem cells (iPS cells)were developed through gene introduction by Prof. Shinya Yamanaka, Japanin 2006, a variety of studies for applying such cells to a therapeuticagent are progressing.

After the report in which stem cells having similar characteristics toembryonic stem cells are established when four genes(dedifferentiation-inducible factors; Oct4, Sox2, c-Myc and Klf4) wereintroduced into mouse or human somatic cells, and then cultured underembryonic stem cell culture conditions for a long time (Cell126:663-676, 2006; Science 318:1917-1920, 2007) had been suggested,various methods capable of replacing genes for clinical use have beenstudied. However, because of still insufficient results of the study onhuman somatic cells, difficulty in defining a dedifferentiation-inducingmechanism, and a risk of forming teratoma, it is difficult to apply iPScells to clinical trials. A direct reprogramming method is a methodwhich has been recently suggested as an alternative for such iPS cells,and includes two types of techniques for introducing genes specificallyexpressed in cells and inducing the cells to desired cells without thepluripotency stage through the regulation of a growth signal bycombination of dedifferentiation-inducible factors and low molecularweight substances. Such a method is highly appreciated in that it hasonly advantages of various stem cells, and eliminates many of thefactors which inhibit a clinical use.

In recent years, the possibility of establishment of mouse OPCs has beenshown by the Marius Wernig research team using three genes (Olig2,Sox10, Zfp536) and by the Paul J Tesar research team using three genes(Olig2, Sox10, NKX6.2) in the United States, and it has been reportedthat they made a success of direct reprogramming from mouse somaticcells to OPCs. However, there is no still report in which human somaticcells are used. In addition, the Shengding and Mickie Bhatia researchteams in the United States demonstrated the establishment of neural stemcells through direct reprogramming by introducing an Oct4 gene into ahuman somatic cell, and thus suggested a new paradigm in which the Oct4gene can regulate the expression of nervous system-related genes incells.

Therefore, the inventors intensively attempted to induce OPCs from humansomatic cells through direct reprogramming, resulting in confirming thepossibility of induction of OPCs by introducing Oct4 into human somaticcells and treating several low molecular weight substances involved informing oligodendrocytes, and thus completed the present invention.

SUMMARY OF THE INVENTION

The present invention is directed to providing a method of inducing OPCsfrom human somatic cells through direct reprogramming, which includesculturing human somatic cells into which a nucleic acid moleculeencoding an Oct4 protein is introduced into a medium containing specificlow molecular weight substances.

The present invention is also directed to providing a method of inducingOPCs from human somatic cells through direct reprogramming, whichincludes culturing human somatic cells in a medium containing specificlow molecular weight substances, wherein the human somatic cells aretreated with an Oct4 protein before, during or after the culture.

The present invention is also directed to providing a composition forinducing OPCs from human somatic cells into which a nucleic acidmolecule encoding an Oct4 protein is introduced or which are treatedwith Oct4 protein through direct reprogramming, the compositionincluding a specific low molecular weight substance as an activeingredient.

The present invention is also directed to providing a method ofdifferentiating the OPCs prepared by the above-described method intooligodendrocytes.

To achieve the above-described objects, the present invention provides amethod of inducing OPCs from human somatic cells through directreprogramming, the method including: culturing human somatic cells, intowhich a nucleic acid molecule encoding an Oct4 protein is introduced, ina medium containing (i) a TGF-β type I receptor inhibitor; (ii) aninhibitor of Rho-associated kinase (ROCK inhibitor); (iii) a histonedeacetylase inhibitor; and (iv) a sonic hedgehog agonist (Shh agonist).

The present invention also provides a method of inducing OPCs from humansomatic cells through direct reprogramming, which includes culturinghuman somatic cells in a medium containing (i) a TGF-β type I receptorinhibitor; (ii) an ROCK inhibitor; (iii) a histone deacetylaseinhibitor; and (iv) a Shh agonist, wherein the human somatic cells aretreated with the Oct4 protein before, during or after the culture.

The present invention also provides a composition for inducing OPCs fromhuman somatic cells into which a nucleic acid molecule encoding an Oct4protein is introduced or which are treated with Oct4 protein throughdirect reprogramming, the composition including (i) a TGF-β type Ireceptor inhibitor; (ii) a ROCK inhibitor; (iii) a histone deacetylaseinhibitor; and (iv) a Shh agonist, as active ingredients.

The present invention also provides a method of differentiating OPCsinto oligodendrocytes, the method including: culturing the OPCs preparedby the above-described method in a medium containing a ROCK inhibitor, acalcium channel agonist and a leukemia inhibitory factor (LIF).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the induction of iOPCs by introducing Oct4 into fibroblastsdifferentiated from Sox10::eGFP hESCs.

FIG. 2 shows the process of selecting a low molecular weight substancenecessary to establish induced OPCs (iOPCs) through culture in inductingmedia (IM) after Oct4-introduced Sox10::eGFP fibroblasts are cultured inreprogramming media (RM) for 7 days.

FIG. 3 shows an FACS analysis result for GFP+ distribution afterOct4-introduced Sox10::eGFP fibroblasts are treated with a low molecularweight substance.

FIG. 4 shows a real-time PCR result for mRNA levels of Olig2, NKX2.2 andZFP536 which are known as markers for OPCs, excluding Sox10, afterOct4-introduced Sox10::eGFP fibroblasts are treated with a low molecularweight substance.

FIG. 5 shows the expression of Olig2, Sox10 and ZFP536 markers, and themorphology of OPCs induced from human somatic cells without usingknockout serum replacement (KSR).

FIG. 6 shows the comparison of Sox10 expression in a medium from whichlow molecular weight substances are removed one by one, and in a mediumcontaining all of the low molecular weight substances.

FIG. 7 shows the induction of iOPCs from human somatic cells withoutusing KSR.

FIG. 8 shows cells undergoing mesenchymal to epithelial transition(MMET) three days after culturing in an inducting medium (IM), and cellswith a similar morphology to OPCs 7 days after culturing.

FIG. 9 shows the expression of PDGFRα and A2B5, which are OPC markers iniPOCs, through FACS analysis.

FIG. 10 shows the expression of PDGFRα and A2B5, in iPOCs, in the cellmembrane, which is identified by immunohistochemical staining.

FIG. 11 shows comparative analysis of mRNA expression of OPC markergenes in iPOCs through real-time PCR.

FIG. 12 shows the differentiation of oligodendrocytes in a typicalbranch type shown when established iOPCs are cultured for 40 to 60 daysunder differentiation conditions in which growth factors are excluded,and an increase in the expression of markers for oligodendrocytes, suchas MBP and MAG, confirmed through real-time PCR and immunohistochemicalstaining.

FIG. 13 shows that iOPCs expressing GFP are co-cultured with mouseneurons and differentiate into oligodendrocytes, and then myelinatedwith the neurons.

FIG. 14 shows myelin obtained after iOPCs are transplanted into amultiple sclerosis animal model, to test in vivo differentiation andefficacy as a therapeutic agent, is similar to that of a normal rat.

FIG. 15 shows the expression of neural stem cell markers such as Sox1,Sox2, and Pax6 genes during an induction period, confirmed throughreal-time PCR to prove that iOPC induction is not achieved bydifferentiation via neural stem cells after Oct4 overexpression.

FIG. 16 shows proliferation after growth factors of OPCs, such as bFGFand PDGF-AA, are removed from a culture solution during induction, toprove that iOPC induction is not achieved via neural stem cells, andiOPCs are proliferated by a reaction with the growth factors, and theexpression of OPC markers such as PDGFRα and A2B5 in the growthfactor-dependent proliferated cells, confirmed by FACS analysis.

FIG. 17 shows the expression of OPC markers after Oct4-overexpressinghair follicle dermal papillae are cultured in IM, which is analyzed byFACS analysis, real-time PCR and immunohistochemical staining.

FIG. 18 shows the expression of OPC markers after Oct4 is overexpressedin amniotic fluid stem cells, adipose-derived stem cells in various ageranges and dermal cells, and cultured in IM, which is analyzed byreal-time PCR or FACS analysis.

FIG. 19 is a diagram showing complete details of the present inventionand the differences from the conventional art.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Unless defined otherwise, all of technical and scientific terms used inthe specification have the same meanings as conventionally understood bythose of ordinary skill in the art to which the present inventionbelongs. Generally, the nomenclature used herein is well known in theart and conventionally used.

In the present invention, it was confirmed that the induction of OPCs ispossibly performed through the introduction of an Oct4 gene treated withvarious low molecular weight substances involved in the generation ofoligodendrocytes after the overexpression of Oct4 in human somatic cellsand the regulation of culture conditions, and also confirmed theexpression of an OPC marker gene, epigenetic characteristics and theability of myelination in vitro were also confirmed. In addition, it wasconfirmed that the OPCs induced thereby are differentiated and thusexhibited efficacy as a cell therapeutic agent.

The present invention relates to the development of a personalized celltherapeutic agent using a direct reprogramming method, and the inventorsdiscovered a novel combination of substances on the basis of lowmolecular weight substances (KR 10-1357402) previously established, andestablished OPCs from human somatic cells by the combination with a genewithout via neural stem cells.

In one exemplary embodiment of the present invention, following theintroduction of an Oct4 gene to foreskin fibroblasts, which are humansomatic cells, the cells were cultured in a medium containing A83-01,thiazovivin, valproic acid (VPA), purmorphamine and forskolin to confirmthe expression of an OPC marker and then induction of OPCs from thehuman somatic cells was confirmed.

Therefore, in one aspect, the present invention relates to a method ofinducing OPCs from human somatic cells through direct reprogramming, themethod including: culturing human somatic cells, into which a nucleicacid molecule encoding an Oct4 protein is introduced, in a mediumcontaining (i) a TGF-β type I receptor inhibitor; (ii) a ROCK inhibitor;(iii) a histone deacetylase inhibitor; and (iv) a Shh agonist.

In one exemplary embodiment of the present invention, although a nucleicacid-type gene encoding an Oct4 protein was introduced into foreskinfibroblasts, which are human somatic cells, to overexpress Oct4, forinduction of OPCs, as well as the introduction of the Oct4 gene into thesomatic cells, direct treatment of the somatic cells with the Oct4protein may be used. The Oct4 protein may be treated before, during orafter the somatic cells are cultured in a medium containing lowmolecular weight substances.

Therefore, in another aspect, the present invention relates to a methodof inducing OPCs from human somatic cells through direct reprogramming,the method including: culturing the human somatic cells in a mediumcontaining (i) a TGF-β type I receptor inhibitor; (ii) a ROCK inhibitor;(iii) a histone deacetylase inhibitor; and (iv) a Shh agonist, where thehuman somatic cells are treated with an Oct4 protein before, during orafter the culture.

In the present invention, the medium may further contain a calciumchannel agonist, or may further contain any one selected from the groupconsisting of RG108, BIX01294, SP600125, lysophosphatidic acid,Bayk8644, forskolin, dexamethasone, EX527 and rolipram, but the presentinvention is not limited thereto.

In the present invention, the TGF-β type I receptor inhibitor may beA83-01, the ROCK inhibitor may be thiazovivin, the histone deacetylaseinhibitor may be valproic acid, the Shh agonist may be purmorphamine,and the calcium channel agonist may be forskolin, but the presentinvention is not limited thereto.

In the present invention, the “A83-01” is a transforming growth factor-βtype I (TGF-β type I) receptor inhibitor, which is a substance thatbinds to a TGF-β type I receptor to interfere with a normal signalingprocess of TGF-β type I (Tojo M et al., Cancer Sci. 96: 791-800, 2005).TGF-β type I is a multifunctional peptide which performs various actionsin cell proliferation and various types of cells, and suchmultifunctionality is known to play a critical role in the growth anddifferentiation of various types of tissue, for example, adipogenesis,myogenesis, bone cell formation, epithelial cell differentiation, andinhibits the proliferation of neural stem cells. In addition to theTGF-β type I receptor inhibitor A83-01, all of the TGF-β type I receptorinhibitors including SB432542 may be used, and the TGF-β type I receptorinhibitor A83-01 may be purchased commercially or prepared to be used asa low molecular weight substance, and the proliferation of neural stemcells is promoted by treatment with the inhibitor. The TGF-β type Ireceptor inhibitor A83-01 is added to the medium so as to be included atan effective concentration. The effective concentration may beinfluenced by parameters well known in the art, such as a medium type, aculture method, etc.

In the present invention, the“n-benzyl-[2-(pyrimidin-4-yl)amino]thiazoie-4-carboxamide (thiazovivin)”is known to block an Rho/ROCK signal inducing apoptosis of neural cellsand neural stem cells and a PTEN signal inhibiting proliferation of theneural stem cells, and expected to inhibit the apoptosis of the neuralstem cells and increase a self-renewal activity and a self-proliferationactivity (Matthias Groszer, et al., Science 294: 2186, 2001).Thiazovivin is a substance for selectively inhibiting a Rho-associatedkinase (ROCK) by an ROCK inhibitor, and may employ Y-27632, etc., inaddition to thiazovivin. Thiazovivin is added to a medium so as to beincluded at an effective concentration, and the effective concentrationmay be influenced by parameters well known in the art, such as a mediumtype, a culture method, etc.

In the present invention, the “2-propylpentaonic acid (VPA)” or“valproic acid (VPA)” is a substance which inhibits a histonedeacetylase, and is known to exhibit a strong cytostatic anticanceractivity by promoting the expression of cell proliferation inhibitoryfactors and genes necessary for inducing differentiation by formingchromatin in a high acetylated state to induce the differentiation ofcancer cells and inhibit angiogenesis, and causing apoptosis of thecancer cells by fixing a cell cycle in the GI state. A histonedeacetylase (HDAC) inhibits gene transcription via pRB/E2F, and thebreakdown in histone acetylation is involved in the generation ofvarious types of cancer. The HDAC is highly expressed under severeenvironmental conditions such as hypoxia, hypoglycemia, cellcarcinogenesis, etc. to promote cell proliferation by inhibiting theexpression of cell proliferation inhibitory factors, and is known to berecognized as a key regulatory factor for cell carcinogenesis anddifferentiation regulation. Particularly, it is known that the VPAinduces inositol reduction, inhibits GSK-3β, activates an ERK pathway,and promotes PPAR activation. Trichostatin (TSA) or a derivative thereofas well as 2-propylpentanoic acid (VPA) may be used as the HDACinhibitor, and the derivative includes various types of pharmaceuticallyacceptable inorganic salts or organic salts. When the treatmentconcentration is too low, there is less of an effect, and when theconcentration is too high, it becomes toxic, and therefore, depending ona cell type, a suitable concentration should be determined.

In the present invention, the “purmorphamine” is a purine compound, andis known to be involved in a Shh signaling system. Purmorphamine is notparticularly limited as long as a Shh signal may be induced, and variousderivatives thereof may be used. For example, commercially available2-(1-naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurin) may be used.Purmorphamine is added to a conventionally used medium to inducededifferentiation into neural stem cell-like cells. In the treatment ofpurmorphamine, which is a Shh derivative, it is advantageous that it isnot necessary to introduce a gene to produce neural stem cells fromhuman fibroblasts. Purmorphamine is to be included at an effectiveconcentration in the medium. The effective concentration ofpurmorphamine may be influenced by parameters well known in the art,such as a medium type and a culture method.

In the present invention, the “forskolin” used herein serves to increasean intracellular cAMP concentration by directly activating a catalyticsubunit of adenylyl cyclase, and the “tranylcypromine” serves to inhibitmonoamine oxidase (MAO), which is an enzyme that normally degradesnorepinephrine at a nerve ending.

In the present invention, the culture medium includes all types of mediaconventionally used in neural stem cell culture, and the medium used inthe culture generally includes a carbon source, a nitrogen source, andtrace elements. The medium of the present invention may be a DMEMcontaining N2, B27, penicillin/streptomycin, non-essential amino acids,bFGF, PDGF and ascorbic acid, but the present invention is not limitedthereto.

As the medium for induced cell culture in the present invention, a basicmedium known in the art may be used without limitation. The basic mediummay be prepared by artificial synthesis, or a commercially availablemedium may be used. Examples of the commercially available media mayinclude, but are not limited to, a Dulbecco's modified Eagle's medium(DMEM), a minimal essential medium (MEM), basal medium eagle (BME), RPMI1640, F-10, F-12, an a-minimal essential medium (a-MEM), a Glasgow'sminimal essential medium (G-MEM), an Isocove's modified Dulbecco'smedium, etc., and the medium is preferably DMEM. In an exemplaryembodiment of the present invention, cells are cultured in DMEM.

In the present invention, the human somatic cells may be, but are notlimited to, foreskin fibroblasts, hair-follicle dermal papillae, IMR90lung fibroblasts or dermal fibroblasts. In addition, OPCs may also beinduced from amniotic-derived stem cells or adipose-derived stem cells,rather than human somatic cells.

Oct4 of the present invention may be provided in the form of a proteinor a nucleic acid encoding the protein, and an Oct4 protein of thepresent invention includes a protein having the amino acid sequence ofthe wild-type Oct4 protein, and a variant of the Oct4 protein.

The variant of the Oct4 protein refers to a protein having a differentsequence due to deletion, insertion, and conservative or conservativesubstitution of one or more amino acid residues, or a combinationthereof, from the natural amino acid sequence of Oct4. The variant maybe a functional equivalent exhibiting the same biological activity asthe natural protein or, if necessary, a variant with an enhancedstructural stability with respect to a physical or chemical environmentor an enhanced physiological activity by changing a physical or chemicalproperty of the protein.

More preferably, the variant is a nucleic acid having the nucleotidesequence encoding the Oct4 protein, and the Oct4-encoding nucleotidesequence is a nucleotide sequence encoding a wild-type or variant-typeOct4 protein. The nucleotide sequence may be modified by substituting,deleting, inserting one or more bases, or a combination thereof, or maybe naturally isolated or prepared by chemical synthesis. The nucleicacid having the nucleotide sequence encoding the Oct4 protein may besingle stranded or a double stranded, and may be a DNA molecule (genomeor cDNA) or an RNA molecule.

The nucleic acid encoding the Oct4 protein may be introduced into cellsby a method known in the art, for example, using naked DNA in a vectorform (Wolff et al. Science, 1990: Wolff et al. J Cell Sci. 103:1249-59,1992), or using a liposome, a cationic polymer or the like. The liposomemay be a phospholipid membrane prepared by mixing a cationicphospholipid such as DOTMA or DOTAP for gene introduction, and a nucleicacid-liposome complex is formed by mixing a cationic liposome and ananionic nucleic acid in a predetermined ratio.

The term “vector” used herein is an expression vector capable ofexpressing a desired protein in suitable host cells, and a geneconstruct including necessary regulatory elements that are operablylinked to express a gene insert.

The term “operably linked” used herein refers to functional linkagebetween a nucleic acid expression regulatory sequence and a nucleic acidsequence encoding a desired protein to perform a general function. Theoperable linkage with a recombinant vector may be formed using a generecombination technique well known in the art, and for site-specific DNAcleavage and linkage, enzymes generally well known in the art may beused.

The vector of the present invention may include a signal sequence orleader sequence for membrane targeting or secretion, as well asexpression regulatory elements such as a promoter, an operator, aninitiation codon, a termination codon, a polyadenylation signal and anenhancer, and may be prepared in various forms according to purpose. Thepromoter for the vector may be constitutive or inducible. Also, theexpression vector may include a selectable marker for selecting hostcells containing a vector, and a replicable expression vector includes areplication origin. The vector may be self-replicated or integrated intohost DNA.

Such a vector may be a plasmid vector, a cosmid vector or a viralvector, and preferably, a viral vector. The viral vector may be, but isnot limited to, a vector derived from a human immunodeficiency virus(HIV), a murine leukemia virus (MLV), an avian sarcoma leukosis virus(ASLV), a spleen necrosis virus (SNV), a Rous sarcoma virus (RSV) or amouse mammary tumor virus (MMTV), an adenovirus, an adeno-associatedvirus, or a herpes simplex virus.

In the present invention, the OPCs may express any one or more markersselected from the group consisting of PDGFR, A2B5, Olig2, Sox10, S100band ZFP536, and may not express Sox1, Sox2 and Pax6 markers.

In still another aspect, the present invention relates to a compositionfor inducing OPCs from human somatic cells into which a nucleic acidmolecule encoding an Oct4 protein is introduced or which are treatedwith Oct4 protein through direct reprogramming, which includes (i) aTGF-β type I receptor inhibitor; (ii) a ROCK inhibitor; (iii) a histonedeacetylase inhibitor; and (iv) a Shh agonist, as active ingredients.

In the present invention, the composition may further contain a calciumchannel agonist, and further contain any one selected from the groupconsisting of RG108, BIX01294, SP600125, lysophosphatidic acid,Bayk8644, forskolin, dexamethasone, EX527 and rolipram.

In the present invention, the TGF-β type I receptor inhibitor may beA83-01, the ROCK inhibitor may be thiazovivin, the histone deacetylaseinhibitor may be valproic acid, the Shh agonist may be purmorphamine,and the calcium channel agonist may be forskolin, but the presentinvention is not limited thereto.

In the present invention, the OPCs may not express the Sox1, Sox2 andPax6 markers.

In another exemplary embodiment of the present invention, it wasconfirmed that induced OPCs differentiate into oligodendrocytes using adifferentiation medium containing triiodo-1-thyronine (T3), thiazovivin,forskolin and LIF, after a growth factor and a specific low molecularweight substance are removed from a conventional induction medium.

Accordingly, in yet another aspect, the present invention relates to amethod of differentiating OPCs into oligodendrocytes, which includesculturing the OPCs prepared by the above-described method in a mediumcontaining a ROCK inhibitor, a calcium channel agonist and LIF.

In the present invention, the medium may be DMEM containing N2, B27,penicillin/streptomycin, non-essential amino acids, ascorbic acid andT3, but the present invention is not limited thereto.

In the case of the OPCs of the present invention which are known ascells that are present in an extremely small amount in the cerebrum,there is a low differentiation rate in neural stem cells constitutingthe cerebrum, and thus there are many difficulties in establishing theOPCs from upper stem cells. Therefore, in the conventional art, whilevarious studies are being conducted to efficiently establish the OPCs,the OPCs can be established through long-term differentiation for atleast 70 days.

The present invention may solve such a problem with a high cellconversion rate in a short period, since the OPCs may be established ina relatively short culturing period such as approximately 1 to 2 weeksthrough direct reprogramming, that is, without via neural stem cells. Inaddition, since there is still no report on the establishment of theOPCs through direct reprogramming using human somatic cells,demyelinating disease-associated therapeutic agents are expected to beuseful in the future cell therapeutic agent market, and the lowmolecular weight substance of the present invention is expected to playa critical role in treating the nervous system.

A demyelinating disease is an intractable neurological disease occurringdue to the absence of oligodendrocytes, and in the conventional art,most studies focus on treating neurological diseases by establishing theOPCs from embryonic stem cells. However, there is difficulty inestablishing the embryonic stem cells as therapeutic agents becausethere is an ethical problem as well as a limit due to immune rejection.Therefore, the method of establishing the OPCs from somatic cellsaccording to the present invention through direct reprogramming maysolve the ethical problem, and since there is no immune rejection, canalso highly contribute to the establishment of personalized celltherapeutic agents.

EXAMPLES

Hereinafter, the present application will be described in further detailwith reference to examples. The examples are merely provided to morefully describe the present application, and it will be obvious to thoseof ordinary skill in the art that the scope of the present applicationis not limited to the following examples.

Example 1: Selection of Low Molecular Weight Substance and Establishmentof Induction Conditions

1-1: Selection of Low Molecular Weight Substance Using Sox10 ReporterSystem

The inventors have established conditions capable of inducing neuralstem cells from mouse cells only with a low molecular weight substancewithout gene introduction (KR 10-1357402). Accordingly, to establish newconditions capable of inducing human somatic cells into OPCs on thebasis of the low molecular weight substance that had been established inthe previous study, a low molecular weight substance was selected usinga Sox10 reporter system (Sox10::eGFP), which is a gene known to beimportant in development of oligodendrocytes (FIG. 1).

When Oct4 gene-introduced cells were cultured in a reprogramming medium(RM: DMEM with high Glucose+5% knock out serum replacement (KSR)+1%penicillin/streptomycin+1% non-essential amino acids+20 ng/ml basic FGF(bFGF)+20 ng/ml human recombinant platelet derived growth factor(PDGF)+50 μg/ml ascorbic acid), one week after induction, it wasconfirmed that a cell length was shorter and a cell size was smallerthrough mesenchymal to epithelial transition (MET), thereby forming anepithelial-like colony. These cells are subcultured in a Matrigel-coateddish, and selected from a medium containing A83-01, thiazovivin,valproic acid and purmorphamine, which is a combination of low molecularweight substances that can induce neural stem cells (DMEM with highglucose+1×N2+1×B27 (without vitamin A)+1% penicillin/streptomycin+1%non-essential amino acids+basic 20 ng/ml FGF (bFGF)+20 ng/ml humanrecombinant platelet-derived growth factor (PDGF)+50 μg/ml ascorbicacid+0.5 μM A83-01+0.5 μM thiazovivin+0.1 mM valproic acid (VPA)+0.5 μMpurmorphamine) by additionally combining an epigenetic modulator and lowmolecular weight substances influencing the development of the nervoussystem.

The conditions for combining low molecular weight substances are asfollows:

ATVP: 0.5 μM A83-01+0.5 μM thiazovivin+0.1 mM valproic acid (VPA)+0.5 μMpurmorphamine, RG108: 0.5 μM RG108, BIX01294: 0.25 μM BIX01294,SP600125: 2 μM SP600125, LPA: 2 μM lysophosphatidic acid, Bayk: 2 μMBayk8644, Fsk: 10 μM forskolin, Dex: 1 μM dexamethasone, EX527: 5 μMEX527, Rolipram: 2 μM rolipram

As a result, FACS analysis showed that Sox10::eGFP was most highlyexpressed (3.31%) under the 10 μM forskolin-addition condition (FIG. 3),and real-time PCR showed that the highest mRNA expression of Olig2,NKX2.2 and ZFP536, which were known as markers for the OPCs, as well asSox10 was also exhibited under the condition (FIG. 4).

1-2: Establishment of KSR-Excluded Induction Conditions

There is a report that it is possible to perform conversion to neuralstem cells when knockout serum replacement (KSR) contained in thereprogramming medium (RM) of Example 1-1 is used with Oct4overexpression.

Therefore, as a result of inducing the OPCs under KSR-excludedconditions to exclude OPCs induced by differentiation, rather thanreprogramming, it was confirmed that the morphology of the OPCsappeared, and the expression of Olig2, Sox10 and ZFP536, which aremarkers for the OPCs, was confirmed by PCR (FIG. 5).

In addition, as a result of removing the treated low molecular weightsubstances one by one under the induction conditions of Example 1-1 toselect the optimum combination of low molecular weight substances, itwas confirmed that the highest Sox10 expression was observed in a mediumcontaining all of the low molecular weight substances (FIG. 6).

Therefore, the established conditions (IM: DMEM with highglucose+1×N2+1×B27 (without vitamin A)+1% penicillin/streptomycin+1%non-essential amino acids+20 ng/ml basic FGF (bFGF)+20 ng/ml humanrecombinant platelet-derived growth factor (PDGF)+50 μg/ml ascorbicacid+0.5 μM A83-01+0.5 μM thiazovivin+250 μM valproic acid (VPA)+0.5 μMpurmorphamine+10 μM forskolin) were determined as induction conditionsfor the OPCs (FIG. 2).

Example 2: Induction and Confirmation of OPCs from Human Somatic Cells

Since the Sox10::eGFP fibroblasts used as the reporter cell line ofExample 1-1 are cells obtained through differentiation from embryonicstem cells (ES cells), other stem cells, excluding fibroblasts, may notbe completely excluded, and thus it may not be seen as directreprogramming from somatic cells. In addition, since a KSR-containingmedium was used in the establishment of neural stem cells after Oct4 wasoverexpressed in human somatic cells by Shengding and Mickie Bhatiaresearch teams in the United States, in this Example, to induce directreprogramming from human somatic cells which are not derived fromembryos without via neural stem cells, which are upper stem cells of theOPCs, a KSR-containing reprogramming medium (RM) was not used (FIG. 7).

That is, as a result of introducing an Oct4 gene into BJ cells,subculturing in a Matrigel-coated dish, and culturing for 4 days underinduction conditions (IM) for the OPCs established in Example 1-2, cellshaving undergone MET were observed, and 7 days after culturing, cellsthat look like oligodendrocytes were observed (FIG. 8).

In addition, 7 days after culturing, to check whether the cells areOPCs, FACS analysis (FIG. 9) and immunohistochemical staining (FIG. 10)showed that representative markers for the OPCs, such as PDGFRα andA2B5, were expressed in approximately 10% of the cells, and real-timePCR showed that markers expressed in OPCs derived from dedifferentiatedstem cells, such as Olig2, Sox10, S100b and ZFP536, were expressed inthe induced OPCs (FIG. 11).

On the basis of the above result, the established cells were namediOPCs.

Example 3: Differentiation of OPCs into Oligodendrocytes

3-1: Differentiation into Oligodendrocytes

To confirm an ability to differentiate OPCs induced in Example 2 intooligodendrocytes, a growth factor was removed from a conventionalmedium, and the medium was replaced with a differentiation medium (DMEMwith high glucose+1×N2+1×B27 (without vitamin A)+1%penicillin/streptomycin+1% non-essential amino acids+50 μg/ml ascorbicacid+40 ng/ml T3 (triiodo-1-thyronine)+0.5 μM thiazovivin+10 μMforskolin+10 ng/ml human leukemia inhibitor factor (LIF)).

As a result, a typical branch-type morphology of the oligodendrocyte wasshown, and real-time PCR showed that MBP and MAG expression increased(FIG. 12).

In addition, as a result of co-culturing with neurons obtained from thehippocampus of a rat to confirm in vitro myelination by differentiatedoligodendrocytes, it was confirmed that the neurons were myelinated bythe differentiated oligodendrocytes (FIG. 13).

3-2: Confirmation of In Vivo Differentiation Activity and TherapeuticActivity

To confirm in vivo differentiation activity and therapeutic activity ofOPCs as a cell therapeutic agent, the OPCs induced in Example 2 weretransplanted into multiple sclerosis animal models (experimentalautoimmune encephalomyelitis mouse model (EAE mouse model)).

As a result, it was confirmed that, while no myelin was shown in aPBS-injected control, in an iOPC-transplanted group, myelin similar tothat in a normal mouse were observed (FIG. 14). In other words, it canbe seen that the iOPCs were differentiated in vivo and thus exhibitedefficacy as a cell therapeutic agent.

Example 4: Induction of OPCs Through Direct Reprogramming

In this Example, to confirm that iOPCs were established through directreprogramming, rather than differentiated from neural stem cells inducedafter Oct4 overexpression during the establishment of iOPCs, a change ingene expression during 7 days of induction was confirmed by real-timePCR.

As a result, it was confirmed that all of the Sox1, Sox2 and Pax6, whichwere known as the markers for the neural stem cells, were not expressedfor 7 days (FIG. 15).

In addition, it was observed that cells were proliferated by FGF2 andPDGF-AA, which were known as growth factors for the OPCs, during 7 daysof induction, and FACS analysis showed that the proliferated cells arecells exhibiting PDGFRα and A2B5 (FIG. 16).

Therefore, based on the above result, it can be proved that iOPCsestablished OPCs through direct reprogramming without via neural stemcells, during 7 days of induction.

Example 5: Induction of OPCs from Various Human Somatic Cells

To confirm whether the induction into OPCs is possibly performed inother human somatic cells as well as the foreskin fibroblasts, fivedifferent types of cells (hair-follicle dermal papillae,amniotic-derived stem cells, IMR90 lung fibroblasts, dermal fibroblastsand adipose-derived stem cells), in which Oct4 was overexpressed, wereinduced into OPCs.

As a result, like the foreskin fibroblasts, the expression of PDGFRα,A2B5 and Olig2, which were representative markers for the OPCs, wasconfirmed by RT-PCR, immunohistochemical staining and FACS analysis(FIGS. 17 and 18).

Therefore, it was proved that the combination of Oct4 and the lowmolecular weight substances is an induction method which can be appliedto various human somatic cells and not an induction method limited to BJcells.

INDUSTRIAL APPLICABILITY

A method of inducing OPCs by treating Oct4-overexpressing human somaticcells with low molecular weight substances according to the presentinvention can establish OPCs with high efficiency in a short period oftime through direct reprogramming without via neural stem cells, andthus is useful as a cell therapeutic agent of an intractabledemyelinating disease.

In the above, specific parts of the present invention have beendescribed in detail. However, it will be apparent to those of ordinaryskill in the art that such detailed descriptions are just exemplaryembodiments, and thus the scope of the present invention is not limitedthereto. Therefore, the actual scope of the present invention will bedefined by the accompanying claims and equivalents thereof.

What is claimed is:
 1. A method of inducing oligodendrocyte precursor cells (OPCs) from human somatic cells through direct reprogramming, comprising: culturing human somatic cells, into which a nucleic acid molecule encoding an Oct4 protein is introduced, in a medium containing (i) a TGF-β type I receptor inhibitor; (ii) an inhibitor of Rho-associated kinase (ROCK inhibitor); (iii) a histone deacetylase inhibitor; and (iv) a sonic hedgehog agonist (Shh agonist).
 2. The method of claim 1, wherein the medium further comprises a calcium channel agonist.
 3. A method of inducing oligodendrocyte precursor cells (OPCs) from human somatic cells through direct reprogramming, comprising: culturing human somatic cells in a medium containing (i) a TGF-β type I receptor inhibitor; (ii) an inhibitor of Rho-associated kinase (ROCK inhibitor); (iii) a histone deacetylase inhibitor; and (iv) a sonic hedgehog agonist (Shh agonist), wherein the human somatic cells are treated with an Oct4 protein before, during or after the culture.
 4. The method of claim 3, wherein the medium further comprises a calcium channel agonist.
 5. The method of claim 1, wherein the medium further comprises any one selected from the group consisting of RG108, BIX01294, SP600125, lysophosphatidic acid, Bayk8644, forskolin, dexamethasone, EX527 and rolipram.
 6. The method of claim 1, wherein the TGF-β type I receptor inhibitor is A83-01, the ROCK inhibitor is thiazovivin, the histone deacetylase inhibitor is valproic acid, and the Shh agonist is purmorphamine.
 7. The method of claim 2, wherein the calcium channel agonist is forskolin.
 8. The method of claim 1, wherein the medium is DMEM containing N2, B27, penicillin/streptomycin, non-essential amino acids, bFGF, PDGF and ascorbic acid.
 9. The method of claim 1, wherein the human somatic cells are selected from the group consisting of foreskin fibroblasts, hair-follicle dermal papillae, IMR90 lung fibroblasts and dermal fibroblasts.
 10. The method of claim 1, wherein the OPCs express any one or more markers selected from the group consisting of PDGFRα, A2B5, Olig2, Sox10, S100b and ZFP536.
 11. The method of claim 1, wherein the OPCs do not express Sox1, Sox2 and Pax6 markers. 